PROJECT SUMMARY This project will investigate mammalian DNA polymerase ? (Pol ?), the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Project 2 (?Biochemical reconstitution and inhibition of TMEJ?) which is part of a Program Project titled, ?Polymerase theta, genome instability, and cancer?. Despite the biological importance of TMEJ, we know surprisingly little about its molecular mechanism and how defects in the process confer specific vulnerabilities in tumors. Pol ? is a large protein (290 kDa in mammalian cells) with a distinctive arrangement of a DNA polymerase domain, a helicase- like domain, and a connecting central domain. This project aims to fill several major gaps in knowledge: First, we will focus on functional domains in Pol ? to analyze the mechanism of DNA microhomology selection and end-trimming. Second, we will define core components that can carry out the TMEJ reaction. Third, we will determine how the genetic background of cancers influences the response to Pol ? suppression, PARP inhibition, and DNA damage sensitivity. In Aim 1 ?Structure-activity analysis of Pol ? in TMEJ?, we will determine the permitted and optimal conditions for end joining, and functional roles of unique Pol ? residues and insertion loops, and candidate nucleases for end-trimming. In Aim 2, ?Function of TMEJ components in a reconstituted reaction?, we will investigate the most biologically relevant DNA ligases roles for PARP and RPA, and we will assess the ability of Pol ? to perform translesion synthesis during joining of DNA tails with damaged bases. In Aim 3, ?Targeting the TMEJ pathway?, we will investigate genetic factors that cause vulnerability in Pol ? defective cells, the influence of PARP inhibitors, and we will begin to explore Pol ? inhibitors. New Pol ? small molecule inhibitors, a Project 2-3 collaboration, will be investigated as research tools. The research work will be highly coordinated within the Program Project with the other three Projects and the three Cores. Our combined diverse approaches include molecular biology, biochemistry, structural biology, and biophysics. Substrates, proteins, and experiments will be designed with Projects 1, 3, and 4 and constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C.